Assay of Transient State Kinetics of RNA Polymerase II Elongation

Abstract

Publisher Summary This chapter outlines an approach for transient state kinetic analysis of elongation by human RNA polymerase II. For human RNA polymerase II, such a complete analysis might not be possible however highly reliable rate data that are informative for inferring essential aspects of mechanism can be obtained. In the presence of elongation factors, specific steps that are targets of regulation can be identified. Currently, this approach is most applicable to factors demonstrated to interact directly with the elongation complex to stimulate, repress, or edit RNA synthesis. Such factors include transcription factor IIF (TFIIF), the TFIIF-interacting component of the CTD phosphatase (FCP1), DRB sensitivity-inducing factor (DSIF), negative elongation factor (NELF), ELL (a leukemia chromosome translocation partner), SIII/elongin, Cockayne syndrome type B protein (CSB), hepatitis delta antigen (HDAg), and SII/TFIIS. So far, the method is validated by analyzing stimulation of RNA polymerase II by TFIIF and HDAg and inhibition by the mushroom toxin α-amanitin. Transient state kinetic analysis is further shown to have utility for analysis of two regulatory human elongation factors, one of which, HDAg, is associated with severe manifestations of hepatitis B infection.